This invention relates to enkephalinase, also known as neutral endopeptidase or kidney brush border neutral proteinase (E.C. 3.4.24.11, recommended name of the Enzyme Commission). The invention further relates to novel forms and compositions thereof and particularly to the means and methods for production of enkephalinase to homogeneity in therapeutically significant quantities. This invention also relates to preparation of isolated deoxyribonucleic acid (DNA) coding for the production of enkephalinase; to methods of obtaining DNA molecules which code for enkephalinase; to the expression of human and mammalian enkephalinase utilizing such DNA, as well as to novel compounds, including novel nucleic acids encoding enkephalinase or fragments thereof. This invention is also directed to enkephalinase derivatives, particularly derivatives lacking cytoplasmic and/or transmembrane portions of the protein, and their production by recombinant DNA techniques.
Enkephalinase has been purified from kidney (Kerr, M. A. and Kenny, A. J. Biochem. J. 137: 477-488 [1974], Gafford, J. et al., Biochemistry 22, 3265-3271 [1983] and Malfroy, B. and Schwartz, J. C., Life Sci. 31, 1745-1748 [1982]), intestine (Danielsen, E. M. et al., Biochem. J. 191, 545-548 [1980]), pituitary (Orlowski, M. and Wilk, S. Biochemistry 20: 4942-4945 [1981]), brain (Relton, J. M. et al., Biochem. J. 215: 755-762 [1983]) and lymph nodes (Bowes, M. A. and Kenny, A. J., Biochem. J. 236: 801-810 [1986]), and has been detected in many peripheral organs (Llorens, C. and Schwartz, J. C., Eur. J. Pharmacol. 69, 113-116 (1981) and in human neutrophils (Connelly, J. C. et al., Proc. Natl. Acad. Sci.[U.S.A.] 82: 8737-8741 [1985]). The distribution of enkephalinase in the brain closely parallels that of the enkephalins. Llorens, C. et al., J. Neurochem. 39: 1081-1089 (1982). Enkephalinase is also present in those peripheral tissues and cells that respond to and/or release various endogenous peptides. Enkephalinase is a membrane-bound glycoprotein with subunit M.sub.r values in the range of 87000 to 94000. Variation in the M.sub.r values are attributed to differences in the extent and pattern of glycosylation.
The substrate specificity of enkephalinase has been studied using the enzyme from rat and human kidney. Malfroy, B. and Schwartz, J. C., J. Biol. Chem. 259: 14365-14370 (1984); Gafford et al., Biochemistry 22: 3265-3271 (1983); and Pozsgay, M. et al., Biochemistry 25: 1292-1299 (1986). These studies indicate that enkephalinase preferentially hydrolyzes peptide bonds comprising the amino group of a hydrophobic residue, shows a marked preference for short peptides, and is most efficient when it acts as a dipeptidyl carboxypeptidase releasing a carboxy terminal dipeptide. Enkephalinase, which had been found in cerebral synaptic membranes, efficiently cleaves the Gly.sup.3 -Phe.sup.4 amide of enkephalins (Malfroy, B. et al., Nature (Lond.) 276: 523-526 [1978]). Enkephalinase has also been found to cleave the heptapeptide (Met.sup.5)enkephalin-Arg.sup.6 -Phe.sup.7 (Schwartz, J. C. et al., In Proceedings International Union of Pharmacology 9th Congress of Pharmacology, 3: ed. by J. F. Mitchell et al., 277-283, McMillan Press Ltd., London, [1984]) as well as a variety of other neuropeptides, such as cholecystokinin (Zuzel, K. A. et al., Neuroscience 15: 149-158 [1985]), substance P (Horsthemke, B. et al. Biochem. Biophys. Res. Comm. 125: 728-733 [1984]), neurotensin (Checler et al., 1983), angiotensin I and angiotensin II (Matsas et al., Biochem J. 223: 433 [1984] and Gafford et al., Biochemistry 22: 3265 [1983]), kinins, e.g. bradykinin (Gafford, J. T. et al., Biochemistry 22: 3265-3271 [1983]), oxytocin (Johnson et al., 1984), and somatostatin (Mumford, R. A. et al., Proc. Natl. Acad. Sci. [U.S.A.] 78:6623-6627 [1981]). While enkephalinase is capable of hydrolyzing many biological peptides in vitro (Kenny, A. J. Trends in Biochem. Sci. 11: 40-42 [1986]), in vivo enkephalinase has to date only been implicated in the hydrolysis of endogenous enkephalins when released in brain (Schwartz, J. C. et al., Life Sciences 29: 1715-1740 [1981] and Lecomte, J. M. et al., J. Pharmacol. Exp. Ther. 237: 937-944 [1986]). Although the levels of enkephalinase in blood are normally very low (Connelly et al., supra) enkephalinase was found to be present in high levels in the serum from patients with adult respiratory distress syndrome (Connelly et al. Supra). Enkephalinase cleaves the chemotactic tripeptide fMet-Leu-Phe. Id. It was also observed that neutrophils from donors who smoked had enkephalinase activites about twice that of nonsmokers. Id. Enkephalinase has also been found in high levels in the microvilli of human placentae (Johnson, A. R. et al., Peptides 5: 789-796 [1984]).
Although the isolation of enkephalinase from various tissues has been described in the literature as shown above, the precise structure of enkephalinase has not been previously established. While some quantities of "purified" enkephalinase have been available as obtained from various tissues, the low concentration of enkephalinase in blood and tissues and the high cost, both economic and of effort, of purifying the protein from tissues makes this a scarce material. It is an object of the present invention to isolate DNA encoding enkephalinase and to produce useful quantities of human and mammalian enkephalinase using recombinant DNA techniques. It is a further object herein to prepare novel forms of enkephalinase. It is still another object herein to provide an improved substrate for the assay of enkephalinase activity. This and other objects of this invention will be apparent from the specification as a whole.